The long term goal of our research is to understand the molecular mechanisms of differentiation in the mammalian nervous system. How a multipotential stem cell segregates daughter cell types of restricted potential with distinct cell fates is a major unsolved problem in developmental biology. The RT4 cell line family is derived from a rat peripheral neurotumor and mimics many aspects of in vivo neuronal and glial differentiation. The putative stem cell line RT4-AC, expresses properties of both neurons and glia and gives rise reproducibly to three derivative cell types. Two of the derivative cell types are neuronal and one is glial. The RT4 derivatives cell types appear to be determined neuronal and glial cell types which are capable of further differentiation under appropriate culture conditions. We are proposing to study cell type-specific gene expression in the RT4 cell line family to 1) identify cell type-specific transcription factors and 2) test the involvement of these factors in cell fate determination in the RT4 system. We are focusing on the conversion of the RT4 stem cell to the neuronal derivative cell types. Identification of neuron-specific transcription factors will be accomplished by two complimentary strategies. The first involves the detailed analysis of the transcriptional regulation of a neuronal marker gene, the voltage-sensitive Na+-channel SkM2. We will clone the 5' flanking genomic regions of the SkM2 gene, construct expression vectors, perform deletion analyses of these constructs to locate cis-acting sequence elements that may bind the transcription factors followed by gel shift and DNase I footprinting analyses to confirm the specificity of binding and better define the sequence element. The cis- acting sequence elements will be used as probes of expression libraries to clone the transcription factor genes. Our second strategy involves examining the expression in the RT4 cell lines of peripheral nervous system-specific transcription factors that have been cloned by others. All transcription factors whose expression segregates in the RT4 system will be examined for their ability to regulate SkM2 expression and for their ability to cause conversion of the RT4 stem cell line. Our studies will contribute to an understanding of the mechanisms underlying cell fate decisions in the mammalian nervous system. In addition, since many diseases are known to affect Na+-channel function our detailed transcriptional analysis of the SkM2 Na+-channel gene will contribute to an understanding of basic neuron function.